Page 1 of 10 Original Article
Expression of HMGB1 and TLR4 in neuropsychiatric systemic
lupus erythematosus patients with seizure disorders
Qin Huang1#, Shuqun Shen2#, Hang Qu3, Yu Huang3, Danni Wu4, Haishan Jiang5, Chao Yuan5,6,7
1Department of Rheumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; 2Dermatology Hospital, 3First Clinical Medicine College, 4Second Clinical Medicine College, Southern Medical University, Guangzhou 510515, China; 5Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China; 6Center for Basic Medical Research & Department of Cardiovascular Surgery, TEDA International Cardiovascular Hospital, Chinese Academy of Medical Sciences, Tianjin 300457, China; 7Postdoctoral Station, Medical College, Nankai University, Tianjin 300457, China
Contributions: (I) Conception and design: C Yuan, H Jiang; (II) Administrative support: None; (III) Provision of study materials or patients: Q Huang, S Shen, H Jiang, C Yuan; (IV) Collection and assembly of data: H Qu, Y Huang, D Wu; (V) Data analysis and interpretation: All authors; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.
#These authors contributed equally to this work.
Correspondence to: Chao Yuan; Haishan Jiang. Department of Neurology, Nanfang Hospital, Southern Medical University, 1838# North Guangzhou Avenue, Guangzhou 510515, China. Email: yuanchao613@163.com; jianghs@smu.edu.cn.
Background: Previous studies show that the high‐mobility group box protein 1 (HMGB1) and the toll‐like receptor 4 (TLR4) participate in systemic lupus erythematosus (SLE). The two molecules contribute to the occurrence and persistence of seizures in various disease conditions, such as epilepsy. Since seizures are one of the most severe complications associated with neuropsychiatric SLE (NPSLE), the current study aimed at investigating whether HMGB1 and TLR4 play any role in NPSLE related seizures.
Methods: Data from 291 SLE patients and 100 healthy controls (HC) were prospectively collected from 2013 to 2018. The ELISA test was used to determine serum levels of HMGB1 for all patients and HC and cerebrospinal fluid (CSF) levels of NPSLE patients. The expression levels of TLR4 by the peripheral blood monocytes (PBMCs) were determined by real-time PCR of TLR4 mRNA. Binary logistic regression and ROC curve analysis were used to predict NPSLE.
Results: Among the 291 SLE patients, 188 had active disease and were grouped into two, NPSLE (N=86) and Non-NPSLE (N=102) groups. Among the NPSLE patients, 21 had seizure disorders. Serum HMGB1 levels were increased in NPSLE (8.73±0.29 ng/mL) and were associated with disease activity (r=0.6527, P=0.000). Both serum and CSF HMGB1 levels in NPSLE patients with seizure disorders (9.59±0.63 and 2.90±2.29 ng/mL, respectively) were higher than in patients with other neuropsychiatric symptoms (8.45±0.33 and 2.56±1.70 ng/mL, respectively), though without significance. The gene expression of mRNA TLR4 in PBMCs was similar to serum HMGB1 in the investigated groups. Independent predictors of NPSLE were SLEDAI-2k (OR 1.25; 95% CI: 1.155–1.353), serum HMGB1 (OR 1.659; 95% CI: 1.266–2.175), and anti-Rib-P Ab (OR 3.296; 95% CI: 1.013–10.725). ROC curves for the above predictors had a large AUC (95% CI) of 0.936 (0.900–0.971), indicating a good prediction of NPSLE occurrence.
Conclusions: The expression of HMGB1 and TLR4 was increased in NPSLE, but HMGB1 and TLR4 had minimal effect on NPSLE related seizures. The serum levels of HMGB1 were positively correlated with disease activity, and could, therefore, be a potential biomarker of NPSLE for use in future clinical practice.
Keywords: High mobility group box 1 (HMGB1); TLR4; neuropsychiatric systemic lupus erythematosus (NPSLE); seizure
Submitted Sep 08, 2019. Accepted for publication Nov 21, 2019. doi: 10.21037/atm.2019.12.44
Introduction
Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by the formation of autoantibodies and inflammation in multiple organs (1). Neuropsychiatric SLE (NPSLE) is a form of SLE associated with severe neuropsychiatric (NP) syndromes, including various neurological and psychiatric features (2). Several studies estimated that between 12% and 95% of their sample SLE patients had NPSLE (1,3-5). The long-term prognosis of SLE patients indicates that seizures are among the most ominous and clinical manifestations of damage accrual by NPSLE (6). However, the pathogenesis of NPSLE related seizures has not yet been elucidated.
The high mobility group box 1 (HMGB1) is a highly conserved nuclear DNA-binding protein released by innate immune cells in pathological conditions (7). Recent studies revealed increased expression levels of serum HMGB1 in SLE patients, indicating that HMGB1 could be a marker of active SLE (8). The toll-like receptor 4 (TLR4) is an endogenous pattern recognition receptor of HMGB1, and it mediates chronic inflammatory and autoimmune diseases (9-12). Also, TLR4 has been shown to facilitate SLE pathogenesis by regulating T cells and inducing autoantibodies (13). Meanwhile, HMGB1 and TLR4 contribute to the occurrence and persistence of seizures. For instance, HMGB1 is an epilepsy-associated cytokine since the pathophysiology of epilepsy indicates that HMGB1 increase in quantity and distribution intensifies seizures. Previous studies observed that susceptibility to seizures was reduced in TLR4 knockout mice (14,15). Using mice models, the study by Mattia et al. revealed that antagonists of HMGB1 and TLR4 retard seizure occurrence and reduced recurrence of acute and chronic seizures (16). However, previous researches on the effects of HMGB1 and TLR4 on seizure disorders involved primary or drug-induced epilepsy, not SLE.
Since HMGB1 and TLR4 participate in both SLE and seizures, it is crucial to determine whether HMGB1 and TLR4 play any role in NPSLE related seizures. Currently, few clinical studies have explored this subject. Besides, two studies examined TLR4 gene polymorphism in NPSLE patients but gave inconsistent conclusions. A study by Bogaczewicz et al. reported no correlation between TLR4 polymorphism and NPSLE in a Polish population (17). On the contrary, a study done in South India reported a positive association between the TLR4 polymorphism T399I and NPSLE related seizures (18). Therefore, this study explored
the expression of HMGB1 and TLR4 in a large group of SLE patients. We evaluated the correlation between disease activity and HMGB1/TLR4 expression, focusing on involvement in neuropsychiatric syndromes, particularly NPSLE related seizures.
Methods
Patients and samples
We prospectively enrolled 291 SLE patients who visited the Department of Rheumatology, Nanfang Hospital, Guangzhou, China, from January 2013 to June 2018. Patients who were older than 14 years and met four of the 1997 revised classification criteria of the American College of Rheumatology (ACR) were eligible for inclusion (19). Patients who had other autoimmune diseases were excluded. The SLE disease activity index 2000 (SLEDAI-2k) was used to assess disease activity patterns in patients (20) with an SLE disease activity of SLEDAI-2k <4 was considered quiescent, while the activity of SLEDAI-2k ≥4 was deemed to be active. The active disease cohort included 188 patients, while the quiescent cohort included 103 patients. Active SLE patients were classified into two groups, NPSLE (N=86) and Non-NPSLE (N=102) groups. The NPSLE definition was based on the 1999 ACR nomenclature and case definitions for neuropsychiatric lupus syndromes (21). Patients who developed neuropsychiatric syndromes not attributable to SLE (electrolyte imbalances, infections or medications) were excluded. Meanwhile, 100 age- and gender-matched healthy controls (HC) were recruited from the Physical Examination Center of Nanfang Hospital.
Clinical data were collected from all patients, and the SLE serologic variables of complement component 3 (C3) and complement component 4 (C4) were recorded. Also recorded were the antibody levels of anti-double-stranded DNA (anti-dsDNA), anti-ribosomal P protein (anti-rib-P), anti-SSA, anti-SSB, anti-cardiolipin (Acl), and anti-β2 glycoprotein I (β2-GPI). All the antibodies were detected at the clinical laboratory of Nanfang Hospital. The neuropsychiatric (NP) syndromes and cerebrospinal fluid (CSF) examination results of NPSLE patients were also recorded.
at −80 ℃ in polypropylene tubes until further use. Blood (3 mL) for RNA extraction was collected in a vacutainer tube containing 15% EDTA solution. Peripheral blood monocytes (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation and stored at −80 ℃ until use for RNA extraction. Cerebrospinal fluid (CSF) samples were collected from the NPSLE patients and stored at −80 ℃ until the biomarker assays were performed.
Measurement of serum and CSF HMGB1
Serum and CSF levels of HMGB1 were determined using an HMGB1 ELISA kit (Shino-Test Corp., Tokyo, Japan) according to the manufacturer’s guidelines. In short, a 96-well plate was coated with samples (10 μL/well) in duplicate and incubated at 37 ℃ for 20 h. The plate was then washed five times in wash buffer and dried by gentle on a lint-free paper towel. Subsequently, 100 μL of the detection antibody solution was added to each well, and the plate was incubated for 2 h at room temperature. An equal volume (100 μL/well) of the substrate solution was then added and incubated at room temperature for 30 min, protected from light. The reaction was stopped by the addition of 100 μL/well of 1 M H2SO4, and the optical density (OD) was determined at a wavelength of 450 nm 5 min later. Results were fitted to the standard curve, and the HMGB1detection range was 0.2–80 ng/mL.
Expression of TLR4 in PBMCs
TLR4 mRNA was extracted from PBMCs using TRizol Reagent (Invitrogen, Carlsbad, CA). The total PBMC samples of 291 patients and 100 samples of HC in each group were divided into several subgroups before RNA extraction. In each subset, a certain amount (1×106 cells) of the PBMC samples were mixed into one. In total, we had 19 PBMC samples of active SLE (NPLSE: n=9; non-NPSLE: n=10), 10 of quiescent SLE, and 10 of the HCs.
Similarly, we divided the PBMC samples of 21 NPSLE with seizure and 65 without seizure into seven subgroups, separately. The concentration of total RNA was quantified using a nanodrop spectrophotometer (Nanodrop 1000; Thermo Scientific). Complementary DNA (cDNA) was synthesized from 200 ng of RNA using random primers and Super-Script II reverse transcriptase (Invitrogen) according to the manufacturer’s instruction. Synthesized cDNAs were used as templates for quantitative real-time PCR analysis using TaqMan gene expression assays (Taq-Man; Applied Biosystems). Relative gene expression levels were determined after normalizing cycle thresholds against the
β-actin housekeeping gene and presented as the relative fold change by the comparative Ct (2–ΔΔCt) method.
Statistical analysis
Data were expressed as means ± standard deviation (SD). Clinical and laboratory variables were compared between groups using the t-test for normally distributed variables, and the Mann-Whitney U test for non-normally distributed data. Differences among observed frequencies were tested using the Chi-square test, while Pearson’s correlation coefficient was used to calculate the correlation between variables. The independent NPSLE variables were predicted by binary logistic regression. The sensitivity and specificity of HMGB1 serum of various patients were determined from the area under the curve (AUC) of the receiver-operating characteristic (ROC) curve and Youden index (YI). A P value <0.05 was considered statistically significant. The GraphPad Prism 7.0a (GraphPad Software Inc., San Diego, CA, USA) and SPSS Statistics 24.0 (IBM Corp, Armonk, NY, USA) software were used for statistical analysis.
Results
Clinical characteristics and laboratory findings of study
objects
[image:3.595.46.286.98.175.2]The clinical characteristics and laboratory findings of the study objects are summarized in Tables 1,2. The mean age of SLE patients (239 females and 52 males) was 33.56±13.10 years. The healthy control (HC) group comprised 100 volunteers (86 females and 14 males), and the mean age was 31.90±12.78 years (Table 1). A total of 188 SLE patients had active disease, while the 103 SLE patients were quiescent. Significant differences were obtained in gender Table 1 Demographic data of population
Variables SLE (n=291) Healthy control (n=100) P value
Age (years, SEM) 33.56±13.10 31.90±12.78 0.272
Sex (F/M) 239/52 86/14 0.373
(P=0.000), anti-SSA antibody (P=0.005), anti-dsDNA antibody (P=0.007), SLEDAI-2k (P=0.000), C3 (P=0.000), and C4 (P=0.000) between the active and quiescent groups of SLE patients. Out of 188 active SLE patients, 86 had neuropsychiatric manifestations. The mean age of NPSLE patients (74 females and 12 males) was 32.81±14.36 years. Levels of anti-dsDNA antibody (P=0.001) and SLEDAI-2k (P=0.000) were significantly higher, while C3 levels were significantly lower (P=0.024) in NPSLE than in non-NPSLE group. Furthermore, anti-rib-P antibody (+) (P=0.012) and anti-SSA antibody (+) (P=0.036) were detected more frequently in NPSLE group samples compared with non-NPSLE group samples. Out of 86 NPSLE patients, 21 had seizure disorders. The occurrence frequency of autoantibodies between the NPSLE with seizure group and without seizure group was comparable. The levels of C3, C4, and SLEDAI-2k between the two groups were equally comparable (Table 2).
The occurrence of NP disease was classified according to the ACR case definitions for NPSLE, including detailed diagnostic guidelines for 19 NP syndromes (21). A total of 14 NP syndromes were identified, and 96 NPSLE events were observed in the 86 NPSLE patients (Figure 1). Among these NP events, central nervous system (CNS) manifestations accounted for 96.5% (83/86 patients), while the involvement of the peripheral nervous system (PNS)
was 15.1% (13/86 patients). The majority of NP symptoms were headache (n=26; 30.2%), seizure disorders (n=21; 24.4%), mood disorders (n=10; 11.6%), myelopathy (n=7; 8.1%) and cognitive dysfunction (n=6; 7.0%).
Serum HMGB1 levels in the investigated groups
Serum levels of HMGB1 in quiescent SLE patients were significantly higher (4.84±0.15 ng/mL) compared to the control group (3.86±0.13 ng/mL), while those in active SLE patients were the highest (7.14±0.19 ng/mL) (Figure 2A). Active SLE patients with NP manifestations showed higher HMGB1 levels (8.73±0.29 ng/mL) compared to active patients without NP manifestations (5.79±0.16 ng/mL) (Figure 2B). Comparable expression levels of serum HMGB1 were found in NPSLE patients with seizure and without seizure disorders (9.59±0.63 and 8.45±0.33 ng/mL, respectively) (Figure 2C).
CSF HMGB1 levels in the NPSLE group
[image:4.595.47.549.93.334.2]The present study obtained 59 CSF samples from NPSLE patients, 21 samples with seizure, and 38 without seizure disorders. The CSF white blood cell count of the two groups was comparable (1.10±1.51 and 2.02±2.23, respectively) (Figure 3A). However, the CSF protein was significantly Table 2 Comparison of clinical and laboratory characteristics between different groups in systemic lupus erythematosus (PSLE) patients
Variables
SLE (n=291) Active SLE (n=188) NPSLE (n=86)
Active (n=188) Quiescent
(n=103) P value NPSLE (n=86)
Non-NPSLE (n=102) P value
With seizure (n=21)
Without seizure (n=65) P value
Sex (F/M) 168/20 71/32 0.000 74/12 94/8 0.236 17/4 57/8 0.476
Age (years) 32.70±13.27 35.14±12.68 0.129 32.81±14.36 32.60±12.35 0.912 35.24±13.31 32.03±14.70 0.377
SLEDAI-2k 19.15±10.39 2.38±1.37 0.000 26.87±9.72 12.64±5.19 0.000 29.62±10.24 25.98±9.46 0.137
Anti-dsDNA (U/mL)
159.42±210.29 100.37±90.26 0.007 215.56±254.56 112.09±149.65 0.001 192.52±248.36 223.00±257.99 0.636
Anti-Rib-P, % 17.6 8.7 0.054 25.6 10.8 0.012 28.6 24.6 0.776
Anti-SSA, % 60.1 42.7 0.005 68.6 52.9 0.036 81.0 64.6 0.188
Anti-SSB, % 19.7 15.5 0.430 18.6 20.6 0.854 14.3 20.0 0.751
Acl, % 8.5 9.7 0.830 9.3 7.8 0.796 4.8 10.8 0.673
β2-GPI, % 4.8 4.9 1.000 5.8 2.7 0.734 4.8 6.2 1.000 C3 (g/L) 0.57±0.31 0.80±0.35 0.000 0.51±0.25 0.62±0.34 0.024 0.50±0.26 0.52±0.25 0.745
higher in the seizure NPSLE group (1.00±0.81 g/L) than in the NPSLE group without seizure disorders (0.41±0.56 g/L) (Figure 3B). A similar expression of CSF HMGB1 was observed in the two groups (2.90±2.29 and 2.56±1.70 ng/mL, respectively) (Figure 3C).
Correlation between serum HMGB1 levels and SLE disease activity
Since HMGB1 is reported to be a potential biomarker for SLE disease activity, this study evaluated the correlation
Aseptic meningitis Acute confusional state Anxiety disorders Guillain Barre syndrome Demyelinating syndrome
Neuropathy, cranial Mononeuropathy
Polyneuropathy Psychoses Cognitive dysfunction
Myelopathy Mood disorders Seizure disorders Headache
[image:5.595.142.476.85.266.2]0 4 8 12 16 20 24 28
[image:5.595.80.525.309.414.2]Figure 1 Distribution of neuropsychiatric syndromes in neuropsychiatric systemic lupus erythematosus (NPSLE) patients.
Figure 2 Serum HMGB1 concentrations in the investigated groups as quantified by ELISA. (A) Serum HMGB1 levels in SLE patients
and healthy controls; (B) serum HMGB1 expression in neuropsychiatric systemic lupus erythematosus (NPSLE) and non-NPSLE patients; (C) comparison of serum HMGB1 in NPSLE with seizure and other NPSLE.
Figure 3 Comparison of cerebrospinal fluid (CSF) white blood cell count (A) CSF protein (B) CSF HMGB1 (C) between neuropsychiatric
systemic lupus erythematosus (NPSLE) patients with seizure and without seizure disorders.
P=0.000 P=0.000
P=0.096 P=0.000
Active Quiescent HC NPSLE non-NPSLE Seizure NPSLE Other NPSLE
12 10 8 6 4 2 0
12 10 8 6 4 2 0
12 10 8 6 4 2 0
HMGB1 ng/mL HMGB1 ng/mL HMGB1 ng/mL
P=0.003
B
C
A
Seizure NPSLE Other NPSLE Seizure NPSLE Other NPSLE Seizure NPSLE Other NPSLE
CSF white blood cell count
CSF pr
otein g/L
CSF HMGB1 ng/mL
P=0.519 P=0.002
P=0.096 10
8
6
4
2
0 10
8 6 4 2 0 −2
4
3
2
1
0
−1
[image:5.595.61.535.485.590.2]between serum HMGB1 levels and various serological parameters. We observed a significant correlation between HMGB1 levels and SLEDAI-2k (r=0.6527, P=0.000) (Figure 4A). We also investigated the correlation between anti-ds DNA antibody and HMGB1 levels. However, no significant correlation was noted between the two parameters (r=0.0430, P=0.465) (Figure 4B). The findings of our study also suggest that serum HMGB1 levels are negatively correlated with C3 (r=−0.2667, P=0.000) and C4 (r=−0.2056, P=0.000) (Figure 4C,D).
The mRNA expression of TLR4 in PBMC in the
investigated groups
The mRNA levels of TLR4 in SLE patients with active disease were higher than those of patients with the quiescent illness, as well as HC (P=0.003 and P=0.000, respectively) (Figure 5A). The mRNA levels of TLR4 were also higher in the NPSLE group compared to the Non-NPSLE group (P=0.000) (Figure 5B). Notably, mRNA expression of TLR4 was comparable between the NPSLE with the seizure group and the group without seizure disorders (Figure 5C).
r=0.6527, P=0.000
r=−0.2667, P=0.000
HMGB1 (ng/mL)
HMGB1 (ng/mL)
HMGB1 (ng/mL)
HMGB1 (ng/mL)
r=−0.2056, P=0.000 r=0.0430, P=0.465 20
15
10
5
0
20
15
10
5
0
20
15
10
5
0
20
15
10
5
0 0 10 20 30 40 50 60
SLEDAI-2k
0.0 0.5 1.0 1.5 2.0
C3 0.0 0.1 0.2 0.3 0.4 0.5C4
0 200 400 600 800 Anti-ds DNA
A
C
B
[image:6.595.121.475.79.325.2]D
Figure 4 Correlations between serum HMGB1 levels and SLEDAI-2k (A), anti-ds DNA (B), C3 (C), and C4 (D) in SLE patients. SLE, systemic lupus erythematosus.
Figure 5 mRNA expression of the TLR4 gene in PBMCs in the investigated groups. TLR4 mRNA expression in active SLE patients (n=19), quiescent SLE patients (n=10) and HC (n=10) (A) neuropsychiatric systemic lupus erythematosus (NPSLE) cases (n=9) and non-NPSLE subjects (n=10) (B) NPSLE with seizure disorders (n=7) and without seizure disorders (n=7) (C).
P=0.000
P=0.000 P=0.204
5
4
3
2
1
0
10
8
6
4
2
0 P=0.003
4
3
2
1
0
P=0.174
Active Quiescent HC
mRNA RQ of TLR4 mRNA RQ of TLR4 mRNA RQ of TLR4
NPSLE Non-NPSLE Seizure NPSLE Other NPSLE
B
C
[image:6.595.60.544.383.500.2]Table 3 Significant variable for neuropsychiatric systemic lupus erythematosus (NPSLE) observed in binary logistic regression
Variables S.E. Wald Sig. Exp(B) 95% CI
SLEDAI-2k 0.040 30.524 0.000 1.250 1.155–1.353
HMGB1 (ng/mL) 0.138 13.439 0.000 1.659 1.266–2.175
Anti-dsDNA (U/mL) 0.001 1.286 0.257 1.001 0.999–1.003
C3 (g/L) 0.804 0.613 0.434 1.878 0.388–9.086
Anti-SSA 0.515 3.674 0.055 2.682 0.978–7.355
Anti-Rib-P 0.602 3.924 0.048 3.296 1.013–10.725
Prediction of NPSLE
Our study results indicate that SLEDAI-2k, HMGB1, anti-ds DNA Ab, C3, anti-SSA Ab, and anti-rib-P Ab are significantly associated with NPSLE. These factors were subjected to binary logistic regression to predict NPSLE. According to the results of the binary logistic regression, variables independently associated with the occurrence of NPSLE include SLEDAI-2k, serum HMGB1and anti-Rib-P Ab. The odds ratios (ORs) of the above independent variables are 1.250 (95% CI: 1.155–1.353; P=0.000), 1.659 (95% CI: 1.266–2.175; P=0.000) and 3.296 (95% CI: 1.013– 10.725; P=0.048), respectively (Table 3).
The ROC curves for serum HMGB1 and other variables in NPSLE are shown in Figure 6. ROC analysis of serum HMGB1, SLEDAI, and anti-rib-p Ab, and their correlation to NPSLE reveals substantial prediction of NPSLE. The above independent variables had an AUC (95% CI) of
0.843 (0.783–0.903), 0.905 (0.862–0.948), and 0.574 (0.491– 0.657), respectively (P<0.05). Notably, ROC analysis for the total risk of the combination of serum HMGB1, SLEDAI, and anti-rib-p Ab had a larger AUC (95% CI) of 0.936 (0.900–0.971) (P<0.0001), indicating an excellent prediction of NPSLE occurrence.
Discussion
The present study reveals three main findings: (I) Serum HMGB1 levels are increased in SLE patients, especially in NPSLE, and therefore correlated with disease activity; (II) the gene expression of TLR4 mRNA in PBMCs is increased in NPSLE and (III) HMGB1 and TLR4 have minimal effect on NPSLE related seizures.
To our knowledge, the present study is the first report to examine the levels of HMGB1 in NPSLE patients. HMGB1 is a crucial gene expressed in the nucleus, and its translocation to the extracellular matrix is a vital warning signal of autoimmune diseases (7). Apart from autoimmune diseases, the expression of HMGB1 is increased in nervous system diseases such as cerebral ischemia, atherosclerosis, and ischemia-reperfusion injury (22-24). Consistent with previous reports (25,26), our study found that HMGB1 concentrations in serum were higher in SLE patients than in healthy controls. Moreover, levels of circulating HMGB1 in SLE patients were positively correlated to SLEDAI-2k and negatively correlated with C3 and C4, suggesting that HMGB1 may be involved in the inflammatory process of SLE. However, no previous study has explored the correlation between HMGB1 and NPSLE. The present study observed that levels of serum HMGB1 are increased in NPSLE patients and are positively associated with disease activity. Our logistic regression and ROC analysis results indicate that serum HMGB1 is independently Figure 6 ROC curve for variables in the prediction of
neuropsychiatric systemic lupus erythematosus (NPSLE).
1.0
0.8
0.6
0.4
0.2
0.0
0.0 0.2 0.4 0.6 0.8 1.0 1−Specificity
Sensitivity
[image:7.595.72.258.236.420.2]related to the occurrence of NPSLE. We postulated that the role of HMGB1 in NPSLE might be based on its common effects on autoimmune and nervous system diseases, such as triggering the release of pro-inflammatory factors and promoting neuronal differentiation of brain cells. However, the specific mechanism needs to be explored further.
The second main finding of the present study is that the mRNA expression of TLR4 in PBMCs was increased in NPSLE. In recent years, studies have shown that TLR4 signaling is a potent trigger for SLE. It was observed that Chaperonin 10, a TLR4 signaling inhibitor can efficiently inhibit cutaneous lupus and lupus nephritis (27,28). Furthermore, compared with TLR4-producing SLE model mice, the titers of anti-nuclear, anti-ds DNA and Acl antibodies were decreased in TLR4-deficient SLE model mice (29). However, few studies have reported on the correlation between TLR4 and NPSLE. Also, only two of these reports examined TLR4 gene polymorphism, and the findings of the two studies were inconsistent because of differences in population dynamics. No correlation was found between CC and CT genotypes of TLR4 1196C/ T and NPSLE in a Polish population. In a South Indian population, however, the TLR4 polymorphism T399I was found to be positively correlated with NPSLE related seizures (17,18).
The last main finding of our study is that HMGB1 and TLR4 have minimal effect on NPSLE related seizures. Seizures are one of the most severe complications of NPSLE and can occur at any time in the course of SLE (30). In the last decade, research interest in the HMGB1-TLR4 pathway in SLE and seizure disorders has seen significant growth separately. An in vitro study demonstrated that the HMGB1-TLR4 pathway upregulates apoptosis in macrophages by triggering nuclear translocation of NF-kB in a MyD88-dependent mechanism. And this immune regulatory pathway plays a vital role in the pathogenesis of SLE (31). The CSF concentrations of HMGB1, IL-6, and IL-17A were increased in patients with autoimmune encephalitis. This increase suggests that CSF HMGB1 could be an essential factor promoting inflammatory responses in autoimmune diseases that involve the CNS (32).
Meanwhile, HMGB1-induced neuronal hyperexcitability on seizure models is mediated by activation of neuronal TLR4 and N-methyl-D-aspartate (NMDA) receptors, which promote calcium influx in pyramidal neuron cell bodies (33,34). Our previous study conducted on 36 NPSLE
patients and 37 Non-NPSLE patients showed increased expression of anti-NMDA receptor antibodies in the NPSLE group (35). In this study, the levels of both serum and CSF HMGB1 in NPSLE with seizure were higher than in patients with other neuropsychiatric syndromes, but without significance. The serum TLR4 mRNA expression levels in PBMCs were similar in the NPSLE with seizure and without seizure group. Although HMGB1-TLR4 participated in both NPSLE and seizures, they had little effect on NPSLE related seizures, which implies different pathophysiology between epilepsy and NPSLE related seizures.
The clinical manifestations of NPSLE are diverse and highly heterogeneous in severity and prognosis, and the precise pathophysiology of NPSLE remains poorly understood. Several immune factors that facilitate NPSLE pathogenesis, such as cytokines and brain-reactive autoantibodies among others, were evaluated. One possible explanation is that the neuronal damage in NPSLE may be caused by the cross-reaction between anti-nuclear antibody and neuronal antigens, resulting from the direct action of anti-neuronal antibodies or immune complex-mediated vasculitis (36). So far, 20 autoantibodies (eleven brain-specific antibodies and nine systemic antibodies) have been identified to be associated with NPSLE (37). In this study, we found that anti-SSA and anti-rib-P antibodies are associated with NPSLE. And anti-rib-P antibody is the independent predictor of NPSLE occurrence. Our findings are consistent with other studies (38,39). However, the antibody occurrence frequency was not significantly different between the NPSLE with seizures and without seizures group. Therefore, which autoantibody contributes to which type of NPSLE is still elusive so far.
Conclusions
This study demonstrated that the expression of HMGB1 and TLR4 was increased in NPSLE, although HMGB1 and TLR4 had minimal effect on NPSLE related seizures. The serum levels of HMGB1 were positively correlated with disease activity, which could be a potential biomarker of NPSLE. Serum HMGB1 can, therefore, be used for clinical practice in the future.
Acknowledgments
Funding: This work was supported by Grant from the National Natural Science Foundation of China (No.81801624), and the grant from Medical Scientific Research Foundation of Guangdong Province of China (No. A2019020).
Footnote
Conflicts of Interest:The authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was in compliance with the Declaration of Helsinki and approved by the ethics committee of Nanfang Hospital, Southern Medical University.
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